Amyotrophic lateral sclerosis (ALS) is a progressive, untreatable, uniformly fatal motor neuron disorder that sometimes develops concurrently with frontotemporal dementia (FTD). ALS is encountered in both sporadic (SALS) and familial (FALS) forms; about 10% of cases are transmitted as autosomal dominant traits. The cause of sporadic ALS is not known. Recently it was discovered that about 30-50% of FALS cases are caused by expansions of a non-coding hexanucleotide G4C2 expansion in the gene C9ORF72. These expansions are also detected in 10-20% of familial FTD, 10% of sporadic FTD and in ~5% of SALS. These statistics define the C9ORF72 G4C2 expansion as the most common cause of ALS. In the present study, we propose to use rAAV type Rh10 to introduce a microRNA to silence expression of the transcripts of C9ORF72 that harbor the offending G4C2 expansion. In Aim 1, we will screen, identify and optimize potential miRNAs in vitro. In Aim 2, we will further characterize the phenotype of our novel C9ORF72mutant transgenic mouse and investigate use of rAAVRh10-C9miRs to silencing the mutant C9 transgene and thereby mitigate the phenotype in this mouse. In Aim 3 we will investigate delivery and efficacy of the rAAVRh10-C9miRs in a non- human primate model as first step to translating this therapy to clinical application. Relevance: We believe that these studies will be highly relevant for several reasons. (1) There is a compelling unmet need for effective ALS treatments; this project focuses on the most common form of FALS, with applicability as well to some cases of SALS and FTD. (2) This investigation will develop the use of intrathecal rAAVRh10 as a gene therapy vector for the CNS; the intrathecal route is advantageous, permitting widespread delivery within the CNS with doses that are an order-of-magnitude lower than are required via intravenous delivery; and minimizing peripheral exposure to virus. (3) rAAV has not been used in human neurodegenerative disorders. The platform we propose to develop here should have broad applicability across a breadth of neurological disorders.